Construction of Unmarked Deletion Mutants in Mycobacteria

Methods Mol Biol. 2009;465:279-95. doi: 10.1007/978-1-59745-207-6_19.

Abstract

Site-specific recombinases such as the Saccharomyces cerevisiae Flp and the P1 phage Cre proteins have been increasingly used for the construction of unmarked deletions in bacteria. Both systems consist of an antibiotic resistance gene flanked by recognition sites in direct orientation and a curable plasmid for temporary expression of the respective recombinase gene. In this chapter, we describe strategies and methods of how to use sequence-specific recombination mediated by Flp and Cre to construct mutants of Mycobacterium smegmatis, Mycobacterium bovis BCG, and Mycobacterium tuberculosis.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • DNA Nucleotidyltransferases / metabolism*
  • Gene Deletion*
  • Integrases / metabolism*
  • Mycobacterium / genetics*
  • Mycobacterium bovis / genetics
  • Mycobacterium smegmatis / genetics
  • Mycobacterium tuberculosis / genetics
  • Recombination, Genetic*

Substances

  • Cre recombinase
  • DNA Nucleotidyltransferases
  • FLP recombinase
  • Integrases